Process for producing thrombin

ABSTRACT

The present invention provides a production method of thrombin which can convert prothrombin into thrombin highly efficiently even in the absence of thromboplastin and blood plasma, easily obtain a starting material for use in the conversion of prothrombin into thrombin, and prepare and purify thrombin in an industrial scale. It is characterized in treating a prothrombin-containing aqueous solution with a Ca salt at 0 to 15° C.

TECHNICAL FIELD

This invention relates to a method for the production of thrombin.

BACKGROUND ART

Thrombin is a serine protease having a molecular weight of about 34,000and an isoelectric point of about 7.1, namely a proteolytic enzyme whichexerts its action at the final stage of the blood coagulation process.That is, it acts upon fibrinogen to form fibrin, thereby generating itsblood coagulation function.

Because of this, thrombin is used clinically as a topical styptic in thesurgical field and as a styptic of upper gastrointestinal bleeding andthe like in the field of internal medicine.

In the living body, thrombin is present in the form of prothrombin asits precursor and formed by receiving limited hydrolysis with activatedfactor X and the like. In general, thrombin is produced by firstlyextracting and purifying prothrombin from human blood plasma as thematerial and then treating the thus purified prothrombin withthroinboplastin and the like. In other words, conversion into thrombinis carried out using purified prothrombin.

Thus, it is the present situation that thrombin is produced throughseveral steps, starting from (1) purification of prothrombin asdescribed above, followed by (2) conversion of prothrombin into thrombinand then (3) purification of thrombin and other necessary steps.

Based on this method, various modification methods have been reported.For example, there are known methods in which blood plasma treated withcitric acid is allowed to contact with an anion exchanger, the thusadsorbed prothrombin is converted into thrombin on said exchanger andthen the converted product is eluted and recovered (U.S. Pat. No.5,143,838, European Patent Publication No. 378798) and in which bloodplasma is treated with low temperature ethanol and then with an anionexchanger, and the thus purified prothrombin is converted into thrombinwhich is subsequently purified by a cation exchanger treatmentUnexamined Published Japanese Patent Application No. 3-128398 (U.S. Pat.No. 5,138,034, European Patent Publication No. 408029)!.

Of these steps, conversion into thrombin is effected for example by amethod in which a snake venom is used or a method in which a highconcentration citric acid salt is used Unexamined Published JapanesePatent Application No. 4-365481 and Unexamined Published Japanese PatentApplication No. 5-194261 (European Patent Publication No. 543178)!, inaddition to the aforementioned method in which thromboplastin is used.Also, Unexamined Published Japanese Patent Application No. 5-186369(European Patent Publication No. 528701) and U.S. Pat. No. 5,143,838describe about conversion of purified prothrombin into thrombin makinguse of calcium chloride.

In this connection, thromboplastin prepared from human placenta ismainly used for the aforementioned purpose, but it cannot always be saidthat it can be obtained in sufficient quantity because of a difficultyin preparing the material. The same problem also occurs in the case ofthe snake venom.

On the other hand, the spontaneous conversion with high concentrationcitric acid salt and the calcium chloride-aided conversion are stilllimited to a laboratory scale level and not established yet asindustrial mass production methods.

In view of the above, the inventors of the present invention haveattempted to develop a method for the industrial scale production ofthrombin and accomplished the present invention as the result.

Accordingly, the object of the present invention is to provide a methodin which the starting material for carrying out conversion ofprothrombin into thrombin can be obtained easily and thrombin can beproduced and purified in industrial scale.

DISCLOSURE OF THE INVENTION

The object of the present invention can be achieved by a method for theproduction of thrombin which comprises treating a prothrombin-containingaqueous solution with a Ca salt at 0 to 15° C.

More illustratively, the thrombin production method of the presentinvention roughly comprises (1) preparation of prothrombin-containingaqueous solution, (2) conversion of prothrombin into thrombin and (3)purification of thrombin.

(1) Preparation of prothrombin-containing aqueous solution

The prothrombin-containing aqueous solution to be used in the presentinvention is not particularly limited, provided that it contains bloodplasma originated prothrombin or prothrombin complex or is a fractioncontaining them. The prothrombin-containing aqueous solution of thepresent invention is prepared using a starting material such as asupernatant fluid obtained by removing cryoprecipitate from blood plasma(de-cryo plasma) or Cohn's I+II+III fraction, II+III fraction, or IIIfraction. Preparation of the prothrombin-containing aqueous solution ofthe present invention from these starting materials can be made by aknown method. For example, it can be made by a method in whichadsorption on an inorganic salt is employed or by an anion exchangertreatment or an affinity chromatography.

According to the present invention, it is desirable to use a prothrombincomplex.

The prothrombin complex to be used herein is a mixture of a bloodcoagulation factor, prothrombin (blood coagulation factor II), withother blood coagulation related factors. Examples of the othercomponents to be included together with prothrombin include bloodcoagulation factors VII, IX, X and the like. It is not necessary to usea highly purified product, and a crude product can be used.

When a crude product is used, it results in an advantage in that thepurification step can be simplified and the cost therefore can bereduced, because it is not necessary to carry out complete purificationof prothrombin prior to its conversion treatment into thrombin.

Origin of the prothrombin complex is not particularly limited. Forexample, a bovine or human origin complex, desirably human origin, maybe used.

Known means can be used for the preparation of a prothrombin complex(-containing aqueous solution). For example, the methods disclosed inUnexamined Published Japanese Patent Application No. 62-10019 andUnexamined Published Japanese Patent Application No. 3-128398 (U.S. Pat.No. 5,138,034, European Patent Publication No. 408029) can be used. Moreillustrative examples include a method in which a prothrombin complex isprepared by treating blood plasma with an anion exchanger and a methodin which a prothrombin complex is prepared using de-cryo plasma obtainedby removing cryoprecipitate from blood plasma.

Examples of the anion exchanger include DEAE base exchangers (forinstance, DEAE-agarose, DEAE-dextran, DEAE-cellulose and the like) andQAE base exchangers (for example, QAE-agarose, QAE-dextran and thelike).

The prothrombin complex obtained by the above means can be subjected toits conversion treatment into thrombin directly as the complex form.

(2) Conversion of prothrombin into thrombin

(2-1) The prothrombin-containing aqueous solution obtained in the abovestep (1) is treated with a Ca salt at 0 to 15° C. The term "to treatwith a Ca salt" as used herein means to add a Ca salt to theprothrombin-containing aqueous solution and carry out its conversioninto thrombin. Illustrative examples of such treatment include a methodin which a Ca salt solution is added to the prothrombin-containingaqueous solution, mixed by stirring and then allowed to stand still, anda method in which a solid Ca salt is directly added to theprothrombin-containing aqueous solution, mixed by stirring and thenallowed to stand still, of which the method in which a Ca salt solutionis added to the prothrombin-containing aqueous solution is preferred.Examples of the Ca salt include calcium chloride, calcium hydroxide,calcium acetate and the like. Its treating concentration isapproximately from 10 to 50 mM. The amount of prothrombin to be used inthe treatment is approximately from 1 to 100 units/ml. The treatingtemperature is preferably from 0 to 10° C. The treating time is about 1to 5 days. In addition, the solution may have a pH value ofapproximately from 6 to 9, preferably from pH 7 to 8.

(2-2) As occasion demands, a known thrombin conversion method may beused in combination. Preferably, the Ca salt treating solution of (2-1)is allowed to undergo autocatalytic reaction in a high concentrationcitric acid salt solution to effect spontaneous conversion intothrombin. The conversion may be carried out at a temperature of from 20to 40° C. for a period of from 1 to 10 days. Examples of the citric acidsalt include sodium citrate and the like. Its concentration may beapproximately from 10 to 40% (w/v). The solution may have a pH value offrom 6 to 9, preferably from 7 to 8.

(3) Purification and other treatments of thrombin

The thus obtained thrombin in the above step (2) is purified by a knownmethod. Examples of the purification method include cation exchangertreatment, ammonium sulfate fractionation treatment, affinitychromatography treatment (for instance, treatment using immobilizedarginyl methyl ester or immobilized heparin) and the like. In thisconnection, further purification may be carried out by dialysis,ultrafiltration, gel filtration and the like known means.

By the above treatment, various types of contaminated protein (forexample, fibrinogen, fibrin, α₁ globulin, α₂ globulin, β globulin, γglobulin, protease and the like) are substantially removed. The thuspurified thrombin may have an approximate specific activity of from 500to 2,000 units/A₂₈₀, preferably from 1,000 to 2,000 units/A₂₈₀.

In this connection, activity of thrombin can be obtained by aconventional measuring method. In a preferred method, it is obtained bymeasuring coagulation time with a BBL fibrometer or the like apparatususing human blood plasma as the fibrinogen source. More illustratively,the activity is obtained by keeping 0.1 ml of a test sample diluted tovarious ratio with physiological saline for 2 minutes at 37° C. and thenmeasuring the coagulation time by adding 0.2 ml of blood plasma kept at37° C. in advance.

Said thrombin is made into pharmaceutical preparations in the usual way.

That is, addition of fillers, heating, sterilization, bacterialfiltration, dispensation in small portions, freeze-drying and the liketreatments may be carried out as occasion demands, as well as otheroptional treatments such as dry heating treatment (Unexamined PublishedJapanese Patent Application No. 62-81327), liquid heating UnexaminedPublished Japanese Patent Application No. 63-243032 (U.S. Pat. No.4,923,815)!, trialkyl phosphate treatment (Unexamined Published JapanesePatent Application No. 60-51116, U.S. Pat. Nos. 4,540,573 and 4,764,369,European Patent Publication No. 131740) and combination of trialkylphosphate treatment with dry heating treatment (Unexamined PublishedJapanese Patent Application No. 3-218322, U.S. Pat. No. 5,151,499,European Patent Publication No. 378208).

Illustratively, the following steps can be employed after the thrombinconversion treatment.

(i) Trialkyl phosphate treatment: The converted thrombin is allowed tocontact with a dialkyl phosphate or trialkyl phosphate having an alkylgroup of 1 to 10, preferably 2 to 10, carbon atoms, particularlypreferably in the presence of a surface active agent, thereby effectinginactivation of viruses (to be referred to as SD treatment hereinafter),and the SD treatment can be carried out in accordance with a knownmethod (Unexamined Published Japanese Patent Application No. 7-33799).Most preferably, the SD treatment is carried out in the presence ofbenzamidines (benzamidine, p-aminobenzamidine and the like), basic aminoacids (arginine, lysine and the like) or ε-aminocaproic acid (EACA).Benzamidines may be added in an approximate amount of from 0.0001 to 0.1M, basic amino acids in an approximate amount of from 0.1 to 1 M, andEACA in an approximate amount of from 1 to 10% (w/v) (each as the finalconcentration).

The surface active agent to be used in the aforementioned SD treatmentis preferably a nonionic surface active agent such as a polyol fattyacid ester, a sorbitan fatty acid ester (a Span compound), apolyoxyethylene-sorbitan fatty acid ester (a Tween compound), apolyoxyethylene-alkylphenol ether (a Triton compound), a polyoxyeLhylenefatty acid ester, a polyoxyethylene higher alkyl ether or the like.Their illustrative examples include Tween 80 (or polysorbate 80), TritonX-100, sulfobetaine and the like.

(ii) Cation exchanger treatment: For example, the method described inBiochem., 10(13), p.2501, 1971, can be used. Examples of the cationexchanger to be used include sulfoethyl types (sulfoethyl-dextran,sulfoethyl-agarose and the like), sulfopropyl types(sulfopropyl-dextran, sulfopropyl-agarose and the like), carboxymethyltypes (carboxymethyl-dextran, carboxymethyl-agarose and the like),Amberlite IRC-50 and the like.

The cation exchanger treatment is carried out under such conditions thatthrombin is isolated as a purified substance from other contaminants.Illustratively, adhesion of thrombin to the cation exchanger and itswashing are carried out preferably using 0.01 to 0.05 M citrate buffer(pH: about 6 to 8) containing 0.01 to 0.1 M sodium chloride, or 0.01 to0.2 M phosphate buffer (pH: about 6 to 8) containing 0.01 to 0.1 Msodium chloride. Also, it is desirable to effect its elution by the useof 0.05 to 0.3 M citrate buffer having an approximate pH value of 6 to8.

(iii) Dry heating treatment: The thrombin solution is subjected to thistreatment as a dry composition by freeze-drying it by a known method.The dry heating treatment is carried out at generally 30 to 100° C.,preferably 55 to 85° C., for generally 3 to 200 hours, preferably 10 to100 hours. In order to protect thrombin from the heat, a knownstabilizing agent may be used, such as a saccharide (monosaccharides,disaccharides), a sugar alcohol (e.g., mannitol), an amino acid(particularly a basic amino acid, e.g., arginine), an organic carboxylicacid or a salt thereof (e.g., sodium citrate), an inorganic salt (e.g.,sodium chloride) or the like.

BEST MODE OF CARRYING OUT THE INVENTION

The following inventive and test examples are provided to furtherillustrate the present invention, though they are not intended as adefinition of the limits of the present invention.

INVENTIVE EXAMPLE 1 Preparation of prothrombin and thrombin

A two liter portion of de-cryo plasma obtained by freeze-thawing humanblood plasma was allowed to contact with a DEAE-dextran (trade name,DEAE-Sephadex A-50; manufactured by Pharmacia Co.) which had beenequilibrated in advance with a buffer solution (pH 7) composed of 0.075M sodium chloride and 0.01 M sodium citrate, and the thus adsorbedprothrombin complex was washed with a buffer solution (pH 7) composed of0.15 M sodium chloride and 0.01 M sodium citrate and then eluted with abuffer solution (pH 7) composed of 1 M sodium chloride and 0.01 M sodiumcitrate. The resulting eluate was dialyzed, mixed with 1/10 quantity of3% calcium chloride and then subjected to 3 days of treatment at 5° C.In this case, a buffer solution (pH 7) composed of 0.75% sodium chlorideand 0.5% sodium citrate was used as the dialysis solvent.

After adjusting to pH 7, to this were added tri-N-butyl phosphate(TNBP), Tween 80 and epsilon-aminocaproic acid (EACA) to respectivefinal concentrations of 0.3% (w/v), 1% (w/v) and 4% (w/v), subsequentlycarrying out 6 hours of incubation at 30° C. to effect inactivation ofviruses.

Next, the above reaction solution was adjusted to pH 6.7 and allowed tocontact with a sulfopropyl-dextran (trade name, SP-Sephadex C-50;manufactured by Pharmacia Co.) which has been equilibrated in advancewith a buffer solution (pH 6.7) composed of 0.04 M sodium chloride and0.02 M sodium citrate, thereby effecting adsorption of thrombin alone.The resulting column was washed with a buffer solution (pH 6.7) composedof 0.04 M sodium chloride and 0.02 M sodium citrate, 0.1 M phosphatebuffer (pH 6.5) and 0.045 M sodium citrate solution (pH 7) in thatorder, and then the adsorbed thrombin was eluted and isolated with 0.1 Msodium citrate solution (pH 7). The resulting eluate was concentratedusing Pericon (nominal molecular weight cutoff of 10,000; manufacturedby Millipore Co.) and passed through a bacterial filter to preparepurified thrombin (at least 1,000 units/A₂₈₀ in specific activity). Thethus obtained thrombin, after adjustment of its ionic strength andactivity, was dispensed in small portions, dry heating and thensubjected to 72 hours or more of freeze drying treatment at 60° C. or80° C.

INVENTIVE EXAMPLE 2

The procedure of Inventive Example 1 was repeated, except thatprothrombin was converted into thrombin by treating the eluate at 5° C.for 1 day in the presence of calcium chloride, in stead of the 3 days oftreatment at 5° C. in the presence of calcium chloride, and subsequentlyadding trisodium citrate to a final concentration of 35% (w/v) andcarrying out 2 days of treatment at pH 8 and at 37° C.

TEST EXAMPLE 1

Relationship between temperature condition and thrombin conversionefficiency at the time of the calcium chloride treatment was examined.The temperature condition of Inventive Example 1 was varied between 5and 37° C. and tie thrombin conversion efficiency was measured undereach condition. In this case, a thrombin conversion efficiency when thecalcium chloride treatment was carried out at 15° C. for 3 hours byfurther adding 1/10 quantity of thromboplastin was defined as 100%. Theresults are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Temp. (°C.)                                                                      Treating time (day)                                                                        Conversion efficiency (%)                              ______________________________________                                        5         3            89                                                     15        3            71                                                     25        3            30                                                     30        2            14                                                     37        2            2                                                      ______________________________________                                    

TEST EXAMPLE 2

Examination was made on the effect of a single Ca salt treatment and acombination of the Ca salt treatment with a high concentration citricacid salt treatment on the thrombin conversion efficiency. The resultsare shown in table 2. In this connection, the criterion of conversionefficiency is as describe in Test Example 1.

                  TABLE 2                                                         ______________________________________                                        Ca salt                 Conversion efficiency                                 treatment   Citrate treatment                                                                         (%)                                                   ______________________________________                                        5° C., 3 days                                                                      none        89                                                    5° C., 1 day                                                                       37° C., 2 days                                                                     89                                                    ______________________________________                                    

COMPARATIVE EXAMPLE 1

Prothrombin was extracted as a simple substance from Cohn's II+IIIfraction with 0.1 M sodium chloride solution. The thus obtained extractwas adjusted to pH 6.7, mixed with 1% calcium chloride (1/10 quantity),thromboplastin (1/10 quantity) and blood plasma (1/40 quantity) and thenincubated at 20° C. for 3 hours. The reaction solution was allowed tocontact with an anion exchanger (SP-Sephadex C-50, manufactured byPharmacia Co.) which has been equilibrated in advance with 0.1 Mphosphate buffer (pH 6.5), and the adsorbed thrombin was eluted with 0.1M citrate buffer (pH 6.7). The resulting eluate was mixed with 0.3%(w/v) TNBP, 1% (w/v) Tween 80 and 4% (w/v) EACA and incubated at 30° C.for 6 hours (SI) treatment). The thus obtained reaction solution wasapplied to a column packed with an immobilized heparin (HeparinToyopearl, manufactured by Tosoh Corporation) which has beenequilibrated in advance with 0.02 M citrate buffer (pH 7.0), andthrombin was eluted with 0.3 M citrate buffer (pH 6.0). The resultingeluate was concentrated using an ultrafiltration membrane (nominalmolecular weight cutoff of 10,000) and passed through a bacterial filterto prepare purified thrombin.

TEST EXAMPLE 3

Properties of the thrombin of the present invention obtained inInventive Example 1 were compared with those of the purified thrombinprepared from Cohn's II+III fraction in Comparative Example 1 and of acommercial thrombin preparation (manufactured by TheChemo-Sero-Therapeutic Research Institute).

(1) Specific activity

Thrombin activity and absorbance at 280 nm of each sample were measuredto calculate and compare its specific activity. As the results, specificactivity of the thrombin of the present invention was 1,000 units/A₂₈₀,while those of the II+III fraction-originated thrombin and thecommercial product were 555 units/A₂₈₀ and 582 units/A₂₈₀, respectively.

(2) Distribution

Results of cellulose acetate electrophoresis showed that β fraction ofthe thrombin of the present invention was 100%, while those of theII+III fraction-originated thrombin and the commercial product were 55%and 92%, respectively.

(3) Molecular weight measurement

When measured by SDS-PAGE electrophoresis (Fast System, manufactured byPharmacia Co.), the thrombin of the present invention was found as asingle band of 34,000 in molecular weight under non-reducing condition,while bands of other than 34,000 in molecular weight were found onhigher and lower molecular weight sides in the II+IIIfraction-originated thrombin and the commercial product.

INDUSTRIAL APPLICABILITY

Prothrombin can be converted into thrombin highly efficiently by theproduction method of the present invention even in the absence ofthromboplastin and blood plasma. Also, according to the productionmethod of the present invention, starting material for use in theconversion of prothrombin into thrombin can be obtained easily andprepared efficiently by a simple procedure, so that cost and labor canbe reduced sufficiently.

Also, employment of the known high concentration citrate treatment(Unexamined Published Japanese Patent Application No. 4-365481) afterthe Ca salt treatment renders possible shortening of the treating period(including Ca salt treatment or citrate treatment) without reducingconversion efficiency of prothrombin into thrombin. The treatment withhigh concentration citrate may be carried out using 10 to 40% (w/v) of acitric acid salt at 20 to 40° C. for 1 to 10 days. Also, the use of aprothrombin complex as the source of prothrombin can save labor forcomplete purification into prothrombin. In addition, the conversionefficiency into thrombin can further be improved by carrying out the Casalt treatment at 0 to 10° C. In consequence, the method of the presentinvention is a useful method suitable for industrial scale production.

We claim:
 1. A method for producing thrombin which comprises treating aprothrombin-containing aqueous solution with a Ca salt at 0 to 15° C. toproduce a calcium salt-treated solution containing thrombin.
 2. Themethod for producing thrombin according to claim 1 wherein said Casalt-treated solution is further treated with 10 to 40% (w/v) of acitric acid salt at 20 to 40° C. for 1 to 10 days.
 3. The method forproducing thrombin according to claim 1 wherein prothrombin is in theform of a prothrombin complex.
 4. The method for producing thrombinaccording to claim 1 wherein the Ca salt treatment is carried out at 0to 10° C.
 5. The method for producing thrombin according to claim 1wherein the Ca salt treatment is carried out for 1 to 5 days.
 6. Themethod for producing thrombin according to claim 1 wherein the Ca salttreatment is a step in which prothrombin is converted into thrombin inthe presence of a Ca salt.
 7. The method for producing thrombinaccording to claim 1 wherein the Ca salt is calcium chloride, calciumhydroxide or calcium acetate.
 8. The method for producing thrombinaccording to claim 1 wherein the Ca salt treatment is carried out in theabsence of thromboplastin and blood plasma.
 9. The method for producingthrombin according to claim 1 wherein the Ca salt treatment is carriedout on a prothrombin complex-containing aqueous solution.
 10. The methodfor producing thrombin according to claim 1 wherein (1) a trialkylphosphate treatment, (2) a cation exchanger treatment and (3) a dryheating treatment are carried out after the Ca salt treatment.
 11. Themethod according to claim 1, wherein the calcium salt-treated solutionthat is produced is a thrombin-containing preparation having a specificactivity of at least 1,000 units/A₂₈₀.